Indução da expressão da Glicerol-3-Fosfato desidrogenase em levedura
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Data
2009-06-18
Autores
Silva, Viviane Cristina [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O gene GPD2 de Saccharomyces cerevisiae, que codifica a enzima glicerol-3- fosfato desidrogenase (G3PDH; EC 1.1.1.8; NAD+: oxidoredutase) foi clonado na levedura Pichia pastoris para expressar extracelularmente a enzima em meio de cultura. Essa enzima apresenta aplicação prática em diversos sistemas acoplados para determinação quantitativa de triacilglicerol, glicerol, ácido fosfatídico e outros fosfolipidios também podendo ser usada para medir atividades enzimáticas em diversos tipos de amostras. Para que a atividade extracelular fosse suficiente em ensaios industriais e biológicos, um estudo de indução da expressão da enzima foi realizado no presente trabalho, que consistiu em escolher o clone que melhor secreta a enzima e estudar o meio de crescimento (BMGY), a densidade inicial celular (0,05 mg/mL), o meio de indução enzimática (BMMY), a natureza do tampão (tampão fosfato), o pH (6,0), o tempo de produção da proteína (4 dias), a concentração da enzima através de membrana filtrante (120 vezes), a melhor fonte de peptona (Acumédia), o estudo de pré-indução celular por estresse osmótico (atividade de 0,477 ± 0,0 U/mL em 24 horas com NaCl 0,35M). O processo de produção da G3PDH mostrou que a máxima produtividade enzimática (795 U/mL e atividade específica de 44,49 U/mg) e biomassa final de 17,75 mg/mL foi obtida com as seguintes condições experimentais: 48 horas de indução com meio BMMY, utilizando 1% de metanol, 1% de glicerol, densidade inicial celular de 0,05 mg/mL, pH 5,0 e sobrenadante concentrado 120 vezes em membrana filtrante.
The GPD2 gene from Saccharomyces cerevisiae, which encodes the enzyme glycerol-3-phosphate dehydrogenase (G3PDH, EC 1.1.1.8, NAD +: oxidoredutase) was cloned in the yeast Pichia pastoris to express the enzyme extracellularly in the culture medium. The enzyme G3PDH has practical application in various systems coupled to quantitative determination of triacylglycerol, glycerol, phosphatidic acid and other phospholipids. It can also be used to measure the enzymatic activities in diverse types of samples. For the application of the enzyme extracellular in industrial and biological tests, a study of induction of expression of the enzyme was accomplished in the present work, that consisted of to choose of clone that more expressing the enzyme, the growth medium (BMGY), the cellular initial density (0.05 mg/mL), the medium of enzymatic induction (BMMY), the buffer nature (phosphate potassium), pH (6.0), the time of production of the protein (4 days), the concentration of the protein (120-fold), the peptone source (Acumédia), the study of pre-induction cellular for osmotic stress (activity of 0.477 ± 0.0 U/mL in 24 hours with NaCl 0.35M). The study of the variable determinative in the process of production of the G3PDH it showed that the maximum enzymatic productivity (0.795 U/mL and 44.49 U/mg of specific activity) and final biomass of 17.75 mg/mL was obtained with the following experimental conditions: 48 hours of induction with medium BMMY, using 1% methanol, 1% glycerol, cellular initial density of 0.05mg/mL, pH 5.0 and the supernatant concentrated 120-fold in filter menbrane.
The GPD2 gene from Saccharomyces cerevisiae, which encodes the enzyme glycerol-3-phosphate dehydrogenase (G3PDH, EC 1.1.1.8, NAD +: oxidoredutase) was cloned in the yeast Pichia pastoris to express the enzyme extracellularly in the culture medium. The enzyme G3PDH has practical application in various systems coupled to quantitative determination of triacylglycerol, glycerol, phosphatidic acid and other phospholipids. It can also be used to measure the enzymatic activities in diverse types of samples. For the application of the enzyme extracellular in industrial and biological tests, a study of induction of expression of the enzyme was accomplished in the present work, that consisted of to choose of clone that more expressing the enzyme, the growth medium (BMGY), the cellular initial density (0.05 mg/mL), the medium of enzymatic induction (BMMY), the buffer nature (phosphate potassium), pH (6.0), the time of production of the protein (4 days), the concentration of the protein (120-fold), the peptone source (Acumédia), the study of pre-induction cellular for osmotic stress (activity of 0.477 ± 0.0 U/mL in 24 hours with NaCl 0.35M). The study of the variable determinative in the process of production of the G3PDH it showed that the maximum enzymatic productivity (0.795 U/mL and 44.49 U/mg of specific activity) and final biomass of 17.75 mg/mL was obtained with the following experimental conditions: 48 hours of induction with medium BMMY, using 1% methanol, 1% glycerol, cellular initial density of 0.05mg/mL, pH 5.0 and the supernatant concentrated 120-fold in filter menbrane.
Descrição
Palavras-chave
Biotecnologia, Pichia pastoris, Glicerol-3-fosfato desidrogenase, Levedura metilotrofica, Methylotrophic yeast, Pichia pastoris, Glycerol-3-phosphate dehydrogenase
Como citar
SILVA, Viviane Cristina. Indução da expressão da Glicerol-3-Fosfato desidrogenase em levedura. 2009. 82 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas, 2009.